See also partitioning in general under “partitioning”

Both polyer/polymer and polymer/salt systems have been used for the purificaiton of antibodies. Using polymer/salt systems, IgG can be partially purified without using any type of ligands. 

Aqueous Two-Phase Systems 

Co-precipitation of non-IgG contaminants with positvely charged polymers parallels the selectivity of anion exchange chromatography. These reagents selectively coprecipitate acidic host cell proteins, DNA and various cell culture additives. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70)

Aqueous two-phase system (ATPS) are formed spontaneously upon mixing two aqueous solutios of structurally different components, such as two polymers or a polymer and a salt, above a certain critical concentration. Extraction of antibodies in an ATPS constitues an interesting alternative to the traditional downstream processing of antibodies, with most of the research utilizing either a polymer-salt system or a functionalized polymer-polymer system. Most of the strategies have relied on enhancing the partitioning of antibodies towards the upper phase (PEG-rich) because most impurities partition preferentally to the bottom phase. (Azevedo, “Chromatography-free recovery of biopharmaceutical through aqueous two-phase processing” Trends in Biotechnology, (2009), 27(4):240-247

PEG/Salt Systems:

PEG/Nacitrate: Azevedo (J Chromatography A, 1213 2008, 154-161) discloses aqueous two phase extraction of antibodies using PEG and sodium citrate, allowing the concentration of the antibodies in the citrate rich phase and removal of most hydrophobic compounds in the PEG rich phase. 

PEG/NaCl/phosphate system: The addition of NaCl to polyethylene glycocl (PEG)/phosphate systems was shown to increase the partition coefficient of IgG from a CHO cell supernatant, from 0.06 to 36.4% (Azevedo, J. Biotechnol. 132, 2007 209). 

Tran (WO/2010/062244) discloses an aqueous two phase extraction precipitation process for antibodies using a forward extration PEG-Phosphate ATPE system in which the antibody is partitioned to the polymer rich phase and then a second ATPE back extraction system by introducing the polymer rich phase form the forward extraction to a new phosphate salt rich phase, cuasing the product to precipitate at the interface between the two phases. 

PEG/Dextran: PEG/dextran systems have been reported fro the purificaiton of IgG in the presence of affinity ligands. The addition of ligands containing carboxylic functionalities has been shown to direct the partition of IgG to the PEG rich phase. 

EOPO Systems:

EOPO-dextran: Aires-Barros (J. Chromatography A, 1995 (2008) 94-100 disclose using aqueous two-phase sytem composed of ethylene oxide/propylene oxide (UCON) and dextran for the purificaiton of IgG from CHO cells supernatant. In UCO/dextran systems IgG partitions preferentailly to the less hydrophobic dextran rich phase. The addition of triethylene glycol-diglutaric acid (TEG-COOh) shifted the IgG partition to the upper phase. Using an ATPS coposed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified in two steps with a global yield of 85% and 88% purity. 

By Hydrophobin – fusion molecules

Joensuu (US 14/902851, published as US 2016-0159854) discloses a method of purifying an antibody by means of a hydrophobin-Protein A fusion and a continous phase separation. In the method a surfactant or detergent is added to the protein liquid whih contains at least one hydropbin to form an extraction solution. Non-ionic surfactants are preferred. Hydrophobins can include HFBI, HFBII, Sc3p, certo-ulmin and cryparin. The mixture is then transferred to a separation vessel and phases are allowed to separate. After intial phase separation, mixed extraction solution is continously added to the separation vessel. Simultaneously a separated strem of the surfactant pahse and a separated stream of the aqueous phase are continsouly removed from the extraction solution. Pferably, the thoroughly mixed extraction solution is pumped into a separation vessel with a first flow speed, in which vessel a surfactn phase and an aqueous phase is allowed to separate. A part of the separated surfactant phase and a part of the aquous phase are continously removed from the separated vessel with a second flow speed, which keeps the volumes of the phase states in the separation vessel unchangeable.